The present invention concerns nucleic acids and proteins for potassium channels, as well as related vectors, host cells, processes for preparation, and methods of use. Included within the present invention are methods of screening for compounds that bind to and/or otherwise modulate the potassium channel proteins disclosed herein. Additionally, the present invention encompasses methods of modulating the potassium channels disclosed herein, for example methods of opening/activating or closing/inactivating said potassium channels.
Among ion channels, potassium ion (K+) channels are the most ubiquitous and diverse. They include three major structural classesxe2x80x94channels with six, four, or two transmembrane domains. The six transmembrane domain potassium channels are divided further into different families, such as Shaker-like, eag-like and Slo-like potassium channels. Recent identification of KvLQT1 established a new family of six-transmembrane potassium channels. Barhanin et al. (1996) Nature 384: 78-80; Sanguinetti et al. (1996) Nature 384: 80-83; Yang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4017-22; Wang et al. (1996) Nature Genetics 12: 17-23. Search of DNA and protein sequence databanks reveals additional potential members of KvLQT1-related channels in C. elegans as well as in the human. Wei et al. (1996), Neuropharmacology 35: 805-29;; Yang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4017-2.
One or more types of K+ channels reside on cell membranes where they are remarkably selective for K+ over other ions. In excitable cells, K+ channels modulate action potential configuration. Efflux of potassium is the major mechanism for repolarization, maintenance, and hyperpolarization of resting membrane potential. Halliwell (1990) in Potassium channels-structure, classification. function and therapeutic potential (N. S. Cook, ed.); 348-381; Jan, L. Y. and Jan, Y. N. (1992), Ann. Rev. Physiol. 54: 537-55; Pongs (1992), Physiol. Rev. 72: S69-S88.
In neurons, K+ channels regulate neuronal excitability, action potential shape and firing pattern, and neurotransmitter release. These channels can be gated by various stimuli, such as intracellular second messengers, membrane potential, ions, and neurotransmitters. Hille (1992), Ionic channels of excitable membranes; Catterall (1995), Ann. Rev. Biochem. 64: 493-531. Neuronal K+ channels are critical to such neuronal functions as neurotransmission and neuroprotection, and they may affect perception, learning, behavior, and the like.
Recently, the nomenclature for KvLQT1 and the KvLQT1-related channels was changed. Biervert et al. (1998), Science 279:403-406. KvLQT1 was re-named KCNQ1, and the KvLQT1-related channels (KvLR1 and KvLR2) were re-named as KCNQ2 and KCNQ3, respectively. Therefore, throughout this specification, reference to KCNQ1 is equivalent to KvLQT1; reference to KCNQ2 is equivalent to KvLR1; and reference to KCNQ3 is equivalent to KvLR2.
Benign familial neonatal convulsions (xe2x80x9cBFNCxe2x80x9d), a class of idiopathic generalized epilepsy, is an autosomal-dominantly inherited disorder of newborns. BFNC has recently been linked to mutations in two putative K+ channel genes, KCNQ2 and KCNQ3. Biervert et al., supra; Charlier et al. (1998), Nature Genetics 18:53-55; Singh et al. (1998) Nature Genetics 18:25-29. Preliminary functional characterization of KCNQ2 confirmed that this gene encodes a voltage-activated K+ channel. Singh et al., supra.
The present invention discloses novel nervous system-specific potassium channels referred to herein as KCNQ2 (formerly called KvLR1) and KCNQ3 (formerly called KvLR2). Within the present invention are human KCNQ2 (FIG. 2), human KCNQ3 (FIG. 23), murine KCNQ2 (FIG. 10), and rat KCNQ2 (FIG. 16 and FIG. 17). The invention encompasses the amino acid sequences of these proteins and the nucleic acid sequences encoding said proteins, as well as variations in the nucleic acid sequences due to degeneracy in the genetic code.
The present invention provides for nucleic acid molecules at least about 70% identical to the consensus sequence of the nucleotide sequences disclosed herein. Preferably, the present invention provides: (a) a purified and isolated nucleic acid molecule encoding a KCNQ2 and/or KCNQ3 protein of the present invention; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 70% sequence identity, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) which will hybridize to (a) or (b) under stringent conditions, said fragment preferably comprising at least 15 nucleotides. Preferred nucleic acid sequences encoding the KCNQ2 and KCNQ3 proteins of the present invention are found in SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:22.
Also within the scope of the present invention are amino acid sequences at least about 70% identical to the consensus sequence of the proteins disclosed herein. Preferably, the invention covers: (a) amino acid sequences comprising the KCNQ2 and/or KCNQ3 proteins of the present invention; and (b) amino acid sequences having at least 70% sequence identity, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a). Preferred amino acid sequences comprising the KCNQ2 and KCNQ3 proteins of the present invention are found in SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:8, SEQ ID NO:6 and SEQ ID NO:23.
The invention further concerns novel nucleic acids and associated vectors, host cells, and methods of use. Preferably, the nucleic acid molecule is a DNA molecule. Further preferred are nucleotide sequences encoding the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:8, SEQ ID NO:18 and SEQ ID NO:27, as well as proteins about 70% or more identical to these sequences. Also preferred are nucleotide sequences about 80% or more identical to SEQ ID NO:1; most preferred are SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:7, SEQ ID NO:17 and SEQ ID NO:26.
The invention further concerns nucleic acids obtained by PCR with degenerate oligonucleotide primers. Persons of ordinary skill in the art could devise such primers based on the consensus sequence described herein. PCR techniques are described in White et al. (1989), Trends Genet. 5: 185-9.
This invention further concerns nucleic acid vectors comprising a nucleic acid sequence coding for a KvLR/KCNQ protein, host cells containing such vectors, and polypeptides comprising the amino acid sequence of a KvLR/KCNQ protein. Preferably, the vector encodes a full-length KvLR/KCNQ protein and the polypeptide is full-length KvLR/KCNQ protein. The inventors prefer frog expression vectors such as pSP64T or derivatives thereof (Melton et al. (1984), Nucl. Acids Res. 12: 7057-70); mammalian cell expression vectors such as pcDNA3 (available from Invitrogen); or bacterial cell expression vectors such as pET-30 (available from Novagen or Promega).
This invention further concerns host cells transformed with the above-described vectors. The inventors prefer Xenopus oocytes, mammalian cells (e.g., HEK293, CHO, L929), and bacterial cells (e.g., E. coli, especially BL21(DE3), available from Novagen). The inventors particularly prefer the cells deposited as ATCC Acc. No. CRL-1573 (American Type Culture Collection, 10801 University Boulevard, Manassas Va. 20110-2209).
The invention also concerns methods for detecting nucleic acids coding for KCNQ/KvLR proteins and processes for detecting molecules that bind to and/or otherwise modulate the activity of KCNQ/KvLR proteins. xe2x80x9cModulatexe2x80x9d encompasses both channel openers/activators and channel closers/inactivators.
The invention also concerns methods of modulating the KCNQ proteins, specifically methods of opening/activating or closing/inactivating the KCNQ2 and/or KCNQ3 channels. Additionally, the present invention encompasses a method of treating disease by modulating the activity of the KCNQ proteins.
All references cited herein, whether supra or infra, are hereby incorporated herein in their entirety.